1. Field of the Invention
The present invention relates to a ubiquitous, nuclear receptor (UR), polynucleotides encoding that receptor, antibodies against that receptor and the use of that receptor in screening assays.
2. Description of the Related Art
Normal growth and differentiation of all organisms is dependent on cells responding correctly to a variety of internal and external signals. Many of these signals produce their effects by ultimately changing the transcription of specific genes. One well-studied group of proteins that mediate a cell's response to a variety of signals is the family of transcription factors known as nuclear receptors. Members of this group include receptors for steroid hormones, vitamin D, ecdysone, cis and trans retinoic acid, thyroid hormone, fatty acids (and other peroxisomal proliferators), as well as so-called orphan receptors, proteins that are structurally similar to other members of this group, but for which no ligands are known. Orphan receptors may be indicative of unknown signaling pathways in the cell or may be nuclear receptors that function without ligand activation. There are indications that the activation of transcription by some of these orphan receptors may occur in the absence of an exogenous ligand and/or through signal transduction pathways originating from the cell surface.
Steroid hormones affect the growth and function of specific cells by binding to intracellular receptors (SR) and forming SR-hormone complexes. SR-hormone complexes then interact with a hormone response element (HRE) in the control region of specific genes and alter specific gene expression. cDNAs for many SRs have been isolated and characterized, making it possible to deduce the amino acid sequences of various steroid/thyroid/retinoic acid receptors and related members of the super family of nuclear receptors (Evans, 1988; Liao et al., 1989; Forman and Samuels, 1990).
Three functional domains have been defined in SRs using a combination of deletion and mutation analysis as well as the construction of chimeric receptors. An amino terminal domain is believed to have some regulatory function. A DNA-binding domain (DBD) has two zinc finger structural elements and recognizes a specific HRE in a responsive gene. Specific amino acid residues in the DNA-binding domain have been shown to confer DNA sequence binding specificity. A hormone-binding-domain (HBD) is at the carboxy-terminal region of the SR. In the absence of hormone, the HBD appears to interfere with the interaction of the DBD with its HRE. Hormone binding seems to result in a conformational change in the SR and relieve this interference. A SR without the HBD constitutively activates transcription but at a low level.
Both the amino-terminal domain and the HBD appear to have transcription activation functions (TAF) that have not been well defined or understood. Acidic residues in the amino-terminal domains of some SRs may be important for these transcription factors to interact with RNA polymerase. TAF activity may be dependent on interactions with other protein factors or nuclear components (Tora et al., 1989; Tasset et al., 1990; Diamond et al., 1990). Certain oncoproteins (e.g., c-Jun and c-Fos) can show synergistic or antagonistic activity with glucocorticoid receptors (GR) in transfected cells. Interaction of the GR with these oncoproteins appears to involve oligo (or di)mer formation through a leucine zipper-like interaction. Receptors for glucocorticoid, estrogen and vitamins A and D have been shown to interact, either physically or functionally, with the Jun and Fos components of AP-1 in the transactivation of steroid- or AP-1 regulated genes (Diamond et al., 1990; Yang-Yen et al., 1990; Owen et al., 1990).
Defects in the expression or mutations in SR genes are responsible for various abnormalities in hormone responses. For example, a defect in the X-chromosome-linked AR has been considered to be responsible for syndromes of androgen resistance, such as testicular feminization (tfm). Mutations in AR genes have been observed in more than 20 individuals with abnormal androgen responses. Mutations in the HBD of AR genes have resulted in changes in one amino acid, introduction of a premature stop codon, deletion of part or all of a domain, or alternative splicing. Such a mutation in AR genes also cause changes in affinities and specificities of the hormone binding and allow mutated AR to utilize other steroid hormones. For this reason, antiandrogens can act as androgens in AR-dependent transactivation of specific genes (Liao et al., 1989; Sai et al., 1990; Kokontis et al., 1991).
Although steroid hormones affect transcription of specific genes, steroid hormones are also known to regulate posttranscriptional processes such as the stabilization or de-stabilization of specific mRNAs. The mechanism by which steroids affect mRNA stability is not known. Intracellular receptor recycling may be involved in steroid hormone mediation of mRNA stabilization and other posttranscriptional effects (Liao et al., 1980; Hiipakka and Liao, 1988).
Some nuclear proteins having a typical three-domain receptor structure but without a known hormonal ligand are called orphan receptors (O'Malley, 1990; Kokontis et al., 1991). Some of these orphan receptors are constitutively active in transactivate target genes without the need to interact with a ligand. It is possible that the functions of some orphan receptors are regulated by binding of natural and/or synthetic compounds to the HBD. These orphan receptors may be useful in finding new hormones or pharmaceutically effective agents.
Based on extensive structure-function studies with receptors for steroid and thyroid hormones, vitamin D and retinoic acids, all nuclear receptors appear to be made up of three separate structural and functional domains (Evans, 1988; Carson-Jurica et al., 1990). The first domain, found in the N-terminal region of the protein, is usually important for gene trans-activation (Godowski et al., 1988; Folkers et al., 1993). This region is poorly conserved in sequence and length among different nuclear receptors and even between the same receptor in different species. How the N-terminal domain participates in gene transactivation is unknown, but interactions with other transcription factors have been proposed.
The second domain is a region adjacent to the N-terminal domain, consists of about 68 amino acids, is rich in basic amino acids, and is responsible for DNA-binding activity (Freedman, 1992). This domain binds two zinc ions, each bound through four sulfur atoms of eight cysteine residues in this domain. The zinc stabilizes secondary structural elements (called zinc fingers or modules) that are important for interaction of the protein with DNA. The sequence of the DNA-binding domain (DBD), although distinct for each receptor type, is highly conserved in all members of this family. In fact, several new nuclear receptors have been discovered, and are part of the present invention. This domain may also participate in the homodimerization of steroid receptors. Specific amino acid residues in the DBD confer DNA sequence-binding specificity (Danielsen et al., 1989; Umesono and Evans, 1989). Nuclear receptors, with the possible exception of the glucocorticoid receptor, appear to reside in the nucleus, even in the absence of ligand. A hinge region connecting the DBD to the C-terminal domain contains a nuclear localization signal. Proteins may interact with this signal sequence to shuttle receptors into nuclei through the nuclear pores. This process appears to be an energy-dependent mechanism for recycling receptors.
The third domain is found in the C-terminus of the protein and is responsible for ligand binding activity. Single amino acid changes (natural or experimentally-induced mutations) in this domain can drastically alter a receptor's binding specificity and ability to modulate gene transcription. This domain also modulates the ability of the receptor to control transcription in the absence of ligand and contains structures important for protein-protein interactions, such as with heat shock proteins and various nuclear receptors. Certain regions of the ligand-binding domain (LBD) are moderately conserved among nuclear receptors, which may reflect conserved function of these elements. In particular, structures called leucine zippers that consist of heptad repeats of leucine and other small hydrophobic amino acids may act as dimerization interfaces for receptor homo- or hetero-dimerization (Forman and Samuels, 1990). Many of the nuclear receptors have been shown to be phosphoproteins, however, the role of phosphorylation in receptor function remains unclear (Power et al., 1991; Lydon et al., 1992; O'Malley and Conneely, 1992).
Nuclear receptors modulate gene expression in target cells by binding to specific DNA HREs usually located upstream of hormonally-regulated genes (Truss and Beato, 1993). An HRE may be either simple or `composite`, where binding sites of other transcription factors overlap or lie adjacent to the HRE (Diamond et al., 1990; Lucas and Granner, 1992). Three classes of simple HREs have been described. The nuclear receptors for androgens, glucocorticoids, mineralocorticoids, and progestins bind inverted repeats of a 6-bp DNA element (AGAACA) separated by three nonconserved base pairs. The consensus sequence for this binding site is 5'-AGAACANNNTGTTCT-3' or (SEQ ID NO:5). The fact that these four different receptors bind to the same response element is consistent with the observation that amino acids in the DBD of these steroid receptors that are critical for DNA recognition are conserved.
Estrogen receptors also bind to a similar 6-bp inverted repeat (AGGTCA) with the consensus sequence 5'-AGGTCANNNTGACCT-3' (SEQ ID NO:6). The palindromic nature of these binding sites led to the hypothesis that steroid receptors bind to DNA as homodimers. This was confirmed by x-ray crystallography of the glucocorticoid receptor bound to its HRE (Luisi et al., 1991). Many of the nonsteroid nuclear receptors (thyroid hormone, retinoic acids, vitamin D, etc.) bind to inverted repeats identical to the estrogen receptor but with different spacing, or to direct repeats with optimal spacing dependent on the particular nuclear receptor. The repeat nature of these binding sites implies that these receptors may bind as head to head or head to tail dimers. A third group of nuclear receptors typified by NGFI-B (also known as NUR 77 or TR3) appear to bind to DNA as a protein monomer that requires only a single half-site for DNA binding (Wilson et al., 1991; Wilson et al., 1993). The sequence of its binding site, 5'-AAAGGTCA-3', is very similar to the half site of the estrogen receptor HRE.
In contrast to steroid receptors, which bind to DNA as homodimers, some members of the nuclear receptor family bind more effectively to DNA as heterodimers. For example, receptors for thyroid hormone (TR), vitamin D (VDR), all trans-retinoic acid (RAR), and peroxisomal proliferators (PPAR) bind to DNA in vitro more strongly and modulate transcription as heterodimers with the 9-cis-retinoic acid (9c-RA) receptor (RXR) (Yu et al., 1991; Kliewer et al., 1992; Leid et al., 1992). Response element specificity for these receptor dimers is complex, somewhat flexible, and dependent on the dimerization partner. Members of this subfamily can bind to direct repeats of the DNA consensus sequence element AGGTCA with variable spacing with the following generalized specificity: 1 bp spacing for RXR:RXR and RXR:PPAR; 2 bp for COUP:COUP; 3 bp for VDR:RXR; 4 bp for TR:RXR; 5 bp for RAR:RXR; and 6 bp for VDR:VDR (Carlberg et al., 1993). Homodimers of RAR and TR and heterodimers of RXR and TR or RAR also function on palindromic repeats with no spacing (Forman and Samuels, 1990).
Initially, it was proposed that a code based on spacing and orientation of half sites could provide transcriptional selectivity to each of these receptor types that bind to the same half site DNA sequence (Naar et al., 1991; Umesono et al., 1991). However, as more response element-receptor dimer combinations are investigated exceptions to the "rule" are appearing. For example, RAR:RXR dimers activate gene expression using response elements with 1- or 2-bp spacing (Durand et al., 1992) and the COUP-TF homodimer recognizes a number of direct repeats with variable spacing while acting as a repressor of gene transcription (Tran et al., 1992). Interactions between TR and RAR have also been documented and may have a role in controlling specific gene transcription (Forman et al., 1989; Forman et al., 1992). Specificity in transcriptional activation may ultimately be determined by a combination of several factors including the relative binding strength of receptor dimers, their relative affinity for a particular response element, and the relative intracellular concentration of receptors and their ligands. The discovery of new receptors will undoubtedly require modifications to present models of control of gene expression by members of this family.
Although nuclear receptors affect the transcription of specific genes by binding to DNA, some hormones are also known to regulate gene expression by posttranscriptional processes, such as altering the stability of specific mRNAs (Liao et al., 1989). The mechanism by which some hormones affect mRNA stability is unclear. It has been suggested that intracellular receptor recycling (Liao et al., 1989; Mendel et al., 1987; Picard et 1990; Rossini and Liao, 1982; Schmidt and Litwack, 1982) may be involved in steroid hormone mediation of mRNA stabilization and other posttranscriptional effects of steroids (Liao et al., 1973; Liao et al., 1980; Liao et al., 1972). Steroid receptor binding of RNA has been described by many investigators (Ali and Vedeckis, 1987; Rowley et al., 1986; Webb and Litwack, 1986). This hypothesis is consistent with the mechanism proposed later for the action of transcriptional factor IIIA (a protein with several zinc fingers) that regulates both the synthesis and stability of 5S RNA (Miller et al., 1985).
Orphan receptors are those member of the nuclear receptor family that do not have a known ligand. Some of these orphan receptors were cloned by taking advantage of the amino acid conservation in the DNA-binding domain of nuclear receptors, and screening cDNA libraries with a hybridization probe derived from this domain. Using this method, orphan receptor cDNAs have been isolated from human testis and prostate cDNA libraries. These orphan receptors were named testis receptor 2 (TR2) (with several isoforms) and TR3. TR3 is the human counterpart of mouse NUR77/N10 (Lau and Nathans, 1987) and rat NGFI-B, which are early response genes induced by nerve or serum growth factors, (Herschman, 1991; Nakai et al., 1990). The brain-specific transcription factor NURR1 is distinct from but related to NUR77 (Law et al., 1992). The estrogen receptor-related receptors, hERR1 and hERR2 were cloned by low stringency hybridization to cDNA libraries with a DNA probe coding for the DBD of the estrogen receptor (Giguere et al., 1988).
Related or different approaches have led to the discovery of other nuclear receptors including multiple isoforms of retinoic acid receptors (Zelent et al., 1989) and thyroid hormone receptors (Lazar, 1993), the peroxisomal proliferator activator receptor (PPAR), and chicken ovalbumin upstream promoter-transcription factor (COUP-TFI, EAR-3), and apolipoprotein AI regulatory protein (ARP-1, COUP-TFII) (Wang et al., 1989). Hepatocyte nuclear factor 4 (HNF-4) is a kidney, liver and intestinal transcription factor that binds to genes for several proteins synthesized in the liver (Sladek, 1990). GF-1/Eryf-1/NF-E1 is a human erythroid transcription factor that binds to many genes expressed in erythroid cells (Honda et al., 1993). SF-1/ELP/Ad4BP are orphan receptors involved in gene expression of various steroid metabolizing enzymes (Lynch et al., 1993). There are indications that the activation of transcription by some of these orphan receptors may occur in the absence of an exogenous ligand (Davis et al., 1991; Kokontis et al., 1991) and/or through signal transduction pathways originating from the cell surface (O'Malley and Conneely, 1992).
The evolutionary relationship of nuclear receptors and other transcription factors is not clear. Given the separate functional and structural domains in the family of nuclear receptors, one possibility is that different domains have independent origins. Another model suggests that nuclear receptors diverged from a single common ancestor (Amero et al., 1992). The precursor probably had multiple domains that initially mediated a simple, signal transduction mechanism, but subsequently acquired increasing complex functions. A phylogenetic tree built from the DNA-binding domain of about three dozen nuclear receptors shows a common precursor of all known nuclear receptors and suggests that these nuclear receptors do not share a common ancestor with other transcription factors, zinc finger proteins, or ligand-binding proteins (Laudet et al., 1992).